MBB222 - Lecture 27: Prokaryotic transcription (pt.2)

what is an operon

a cluster of genes in bacteria and other simple organisms (not all prokaryotes) that are controlled by a single on and off switch and are transcrible together into one piece of messenger RNA → polycistronic mRNA

• controlled by a single promoter - allows a cell to control, copy, coordinate multiple related genes a the same time

what is a polycistronic mRNA

each gene on the polycistronic mRNA can be translated independently

• amount of proteins curated can vary between each gene

• transcript terminators may exist at the end of genes → can vary

  • strong transcription terminators (DNA stop sequences) can stop transcription completely while other transcription terminators stall transcription

what is the lac operon

set of genes in bacteria (i.e. e.coli) that are responsible for the uptake / breakdown of lactose

what is E.coli’s preferred source for carbon and energy

glucose

• can use lactose - but has to hydrolyze it to galactose and glucose by using beta-galactosidase (an enzyme that is produced in low levels)

what does allolactose do

ix an isomer of lactose - an inducer of the lac operon

• binds to the lac repressor protein - changes its shape → stops it from blocking the genes required to break down lactose

what are the 3 structural genes of lac operon

• lac Z

• lac Y

• lac A

how does the lac operon generally work

• genes are normally expressed at low levels → repressed

• when lactose is present - they are induced → expression is high

• genes are still expressed at high levels when lactose is present and glucose is absent

regulation ensures that beta-galactosidase is produced only when needed (when lactose is present and glucose is absent)

what is positive regulation

turns genes on using an activator protein

what is a negative regulation

turns genes off by using an repressor protein

how does an adjacent lac operon repress the production of lactose digesting enzymes

• adjacent gene codes for the lac repressor protein (Lacl)

• when lactose is absent - lac repressor binds to lac operator → overlaps the promoter → blocks transcription → stops the cell from making lactose digesting enzymes

describe the negative regulation of the lac operon (how does it turn off)

when lactose is absent → lac operons is in repressed state by Lacl repressor bound to the operator

when lactose is present → conversion of glucose and galactose oocurs by beta-galactosidase

beta-galactosidase can isomerize some lactose → allolactose ( an effector that induces expression of the lac operon)

• allolactose binds to the lac repressor → induces a conformation change → repressor dissociates from the operator and prevents additional repressor from binding to the lac operon

what is the positive regulation of the lac operon

lac operon has a weak promoter (low efficiency) even if allolactose / lactose is preset → transcription does not occur in high levels

expression of the lac operon is activated when glucose is absent

what is the cAMP receptor protein (CRP)

an activator for the lac operon → positive regulator

CRP binds to the effector cAMP → changes it’s conformation and substantially increases it’s affinity for the lac promoter

• CRP - cAMP binding to lac promoter stimulates transcription by directly binding to RNA polymerase

• CRP - cAMP binding distorts the DNA → allows DNA polymerase to bind more effectively

what is cAMP

produced by adenylate cyclase (an enzyme)

• adenylate cyclase is indirectly inhibited by glucose

high levels of glucose → cAMP levels are low - not an effective activator (catabolite repression)

low levels of glucose → cAMP is made - levels are high

what happens when no lactose is present

lac operon is switched off - no lac mRNA is synthesized regardless of glucose levels

what happens when lactose is present / glucose is absent

inducer allolactose is produced - binds to lac repressors → inactivates it

no glucose → high levels of cAMP

cAMP binds to CRP → binds to lac promoter → stimulates transcription

what happens when lactose is present and glucose is present

inducer allolactose is produced and binds to lac repressors and inactivates it

glucose is present → low levels of cAMP → CRP - cAMP doesn’t stimulate transcription

only low level of transcription occurs → beta-galactosidase is not needed as glucose is present (preferred source)

what is trp operon

5 genes needed in order to synthesize amino acid tryptophan (Trp)

• only transcribed when Trp is needed

• when external Trp are high - Trp binds to the Trp repressor protein → binds to the trp operator → represses transcription

• when Trp levels are low - repressor does not bind → Trp operon is transcribed (example of feedback inhibition)

what is feedback inhibition

cellular control mechanism where the end product of a metabolic pathway inhibits na enzyme from an earlier step

how does the binding of Trp repress replication

• Trp binding induces a conformation change in Trp repressor → enables it to bind tightly to trp operator

• Trp repressor dimer binds the Trp operator via helix-turn-helix motifs → operator overlaps with the promoter → binding of the Trp repressor prevents RNA polymerase from the promoter